Structure of the Month: February 2009 [see all]
Structural basis for catalysis of a tetrameric, class IIa fructose 1,6-bisphosphate aldolase from M. tuberculosis
Scott D Pegan†, Kamolchanok Rukseree‡,§, Scott G Franzblau‡, Andrew D Mesecar†
†Center for Pharmaceutical Biotechnology and
‡Institute
for Tuberculosis Research, University of Illinois at Chicago, 60612
§National Center for Genetic Engineering and Biotechnology (BIOTEC),
NSTDA, Thailand Science Park, 12120
Mycobacterium tuberculosis, the causative agent for Tuberculosis (TB), currently infects one third of the world's population in its latent form1. New cases of active TB are on the rise, and the emergence of multidrug resistant strains, MDR-TB and XDR-TB, has highlighted the need for new pharmacological targets within M. tuberculosis2. Studies have proposed Class IIa fructose 1,6-bisphosphate aldolases of M. tuberculosis (MtFBA) as one of these targets. Class II FBAs are critical for bacterial, fungal and protozoan glycolytic pathways due to their ability to catalyze the reversible enol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to fructose 1,6-bisphosphate (FBP)3. Higher organisms lack this type of aldolase, leading to its suggested use as a therapeutic target. Inhibitors of FBAs have been previously developed but lack specificity4. Additionally, a lack of structural characterization of substrate-bound class II FBAs is currently an obstacle to structure-based drug design3.
To begin addressing this hurdle and to determine the tetramerization mechanism of class IIa FBAs, we solved the high-resolution X-ray structures of MtFBA bound to dihydroxyacetone phosphate (DHAP) and fructose 1,6-bisphosphate (FBP) using 22-ID at SERCAT, Advanced Photon Source (APS), Argonne National Labs. Through these structures we observed a tetrameric MtFBA in complex with a mixture of DHAP and hydroxy enolate intermediate (HEI) as well as FBP in the MtFBA-FBP X-ray structure (Figure 1,2).
|
|
|
Figure 1: Cartoon representation of the biological tetramer of MtFBA. The tetramer is created by the asymmetric protomer of MtFBA (orange) and three crystallographic symmetry related protomers (gray, pink, blue). Sodium (purple) and zinc (hot pink) are represented as balls corresponding to their respective atomic radii. Substrates are omitted. |
The appearance of intact FBP in the MtFBA active site, suggested that crystallized MtFBA may be catalytically hindered by the low pH, 4.8, of the crystallization conditions. This provided us a unique opportunity to elucidate a precatalysis structure in which both DHAP and the second substrate G3P were bound within the active site. Using our in-house Rigaku RUH2R generator with VariMax HR optics and R-AXIS IV++ detector, we were able to obtain a complete data set to 2.1 Å for the MtFBA-DHAP crystal soaked in G3P (Table 1). Molecular replacement with MtFBA-DHAP, minus the DHAP, and subsequent refinement with DHAP/HEI and G3P present provided for clear 2Fo-Fc map density of both substrates (Figure 2). As a result, for the first time amongst FBAs, the G3P binding pocket could be defined and the orientation of G3P prior to catalysis observed. This directly led to a refinement of the reaction mechanism for MtFBA and suggested residues that may be involved in properly orienting G3P for the enolate condensation of the reaction.
Through the structure MtFBA-DHAP-G3P, elucidated on our in house Rigaku system, and other structures obtained at APS, the binding sites of G3P, DHAP, HEI, and FBP were defined. In addition, these structures uncover the unique tetrameric composition of a class IIa FBA. The molecular details revealed by those high-resolution structures provide new structural information for refining class II FBA reaction mechanism.
|
|
|
Figure 2: Active site of MtFBA with bound substrates. (A) Asymmetric protomer MtFBA (orange) bound with a mixture of DHAP (light blue) and HEI (green). Zinc (hot pink) and sodium (purple) are rendered in space filling according to their respective atomic radius. Dimeric symmetry-related MtFBA is shown in gray. The 2Fo-Fc map density calculated from the MtFBA-DHAP crystal with DHAP and HEI present is contoured (blue) at 1σ. (B) Asymmetric protomer MtFBA (orange) bound with a mixture of DHAP (light blue) and HEI (green) as well as G3P (yellow). Zinc, sodium, and symmetry-related MtFBA are rendered as in (A). The 2Fo-Fc map density calculated from the MtFBA-DHAP-G3P crystal with DHAP and HEI present is contoured (blue) at 1σ. (C) Asymmetric protomer MtFBA (orange) bound with FBP (pink). Zinc, sodium, and symmetry-related MtFBA are rendered as in (A). The Fo-Fc map density calculated from the MtFBA-FBP crystal without FBP present is contoured (green) at 3σ. |
This work was performed in the laboratory of Andrew D Mesecar at the Center for Pharmaceutical Biotechnology, University of Illinois at Chicago.
Data collection details
|
Sample |
MtFBA-DHAP-G3P |
|
PDB ID |
|
|
Space group |
I222 |
|
Unit cell |
a = 60.8 Å; b = 119.2 Å; c = 164.2 Å |
|
Crystal size |
0.4 mm |
|
Radiation |
Cu Kα |
|
Generator |
RUH2R |
|
Optic |
|
|
Detector |
|
|
Low temperature system |
X-stream 2000 |
|
Crystal-to-detector distance |
165 mm |
|
Exposure time per frame |
15 min |
|
Oscillation width |
1° |
|
Number of frames |
154 |
Resolution range |
96.7-2.1 Å |
References
- World Health Organization. (2008). Global tuberculosis control -surveillance, planning, financing.
- World Health Organization. (2008). Tuberculosis MDR-TB & XDR-TB. February.
- Pegan, S. D., Rukseree, K., Franzblau, S. G. & Mesecar, A. D. (2009). Structural Basis for Catalysis of a Tetrameric Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis. J Mol Biol.
- Fonvielle, M., Weber, P., Dabkowska, K. & Therisod, M. (2004). New highly selective inhibitors of class II fructose-1,6-bisphosphate aldolases. Bioorg Med Chem Lett 14, 2923-6.